This invention relates to novel pharmaceutically useful compounds, in particular competitive inhibitors of trypsin-like serine proteases, especially thrombin, their use as medicaments, pharmaceutical compositions containing them and synthetic routes to their production.
Blood coagulation is the key process involved in both haemostasis (i.e. the prevention of blood loss from a damaged vessel) and thrombosis (i.e. the formation of a blood clot in a blood vessel or in the heart, sometimes leading to vessel obstruction).
Coagulation is the result of a complex series of enzymatic reactions. One of the ultimate steps in this series of reactions is the conversion of the proenzyme prothrombin to the active enzyme thrombin.
Thrombin is known to play a central role in coagulation. It activates platelets, leading to platelet aggregation, converts fibrinogen into fibrin monomers, which polymerise spontaneously into fibrin polymers, and activates factor XIII, which in turn crosslinks the polymers to form insoluble fibrin. Furthermore, thrombin activates factor V and factor VIII leading to a xe2x80x9cpositive feedbackxe2x80x9d generation of thrombin from prothrombin.
By inhibiting the aggregation of platelets and the formation and crosslinking of fibrin, effective inhibitors of thrombin would therefore be expected to exhibit antithrombotic activity. In addition, antithrombotic activity would be expected to be enhanced by effective inhibition of the positive feedback mechanism.
The development of low molecular weight inhibitors of thrombin has been described by Claesson in Blood Coagul. Fibrinol. (1994) 5, 411.
Blombxc3xa4ck et al. (J. Clin. Lab. Invest. 24, suppl. 107, 59, (1969)) reported thrombin inhibitors based on the amino acid sequence situated around the cleavage site for the fibrinogen Axcex1 chain. Of the amino acid sequences discussed, these authors suggested the sequence Phe-Val-Arg (P9-P2-P1, hereinafter referred to as the P3-P2-P1 sequence) would be the most effective inhibitor (for a classification of substrate specificity see Schechten and Bergen, Biophys. Res. Commun. (1967) 27, 157 and (1968) 32, 898).
Thrombin inhibitors based on dipeptidyl derivatives with an xcex1,xcfx89-aminoalkyl guanidine in the P1-position are known from U.S. Pat. No. 4,346,078 and International Patent Application WO 93/11152. Similar, structurally related, dipeptidyl derivatives have also been reported. For example International Patent Application WO 94/29336 discloses compounds with, for example, aminomethyl benzamidines, cyclic aminoalkyl amidines and cyclic aminoalkyl guanidines in the P1-position; European Patent Application 0 648 780, discloses compounds with, for example, cyclic aminoalkyl guanidines in the P1-position.
Thrombin inhibitors based on peptidyl derivatives, also having cyclic aminoalkyl guanidines (e.g. either 3- or 4-aminomethyl-1-amidinopiperidine) in the P1-position, are known from European Patent Applications 0 468 231, 0 559 046 and 0 641 779.
Thrombin inhibitors based on tripeptidyl derivatives with ie aldehyde in the P1-position were first disclosed in European Patent Application 0 185 390.
More recently, arginine aldehyde-based peptidyl derivatives, modified in the P3-position, have been reported. For example, International Patent Application WO 93/18060 discloses hydroxy acids, European Patent Application 0 526 877 des-amino acids, and European Patent Application 0 542 525 O-methyl mandelic acids in the P3-position.
Inhibitors of serine proteases (e.g. thrombin) based on electrophilic ketones in the P1-position are also known. For example, European Patent Application 0 195 212 discloses peptidyl xcex1-keto esters and amides, European Patent Application 0 362 002 fluoroalkylamide ketones, European Patent Application 0 364 344 xcex1,xcex2,xcex4-triketocompounds, and European Patent Application 0 530 167 xcex1-alkoxy ketone derivatives of arginine in the P1-position.
Other, structurally different, inhibitors of trypsin-like serine proteases based on C-terminal boronic acid derivatives of arginine and isothiouronium analogues thereof are known from European Patent Application 0 293 881.
More recently, thrombin inhibitors based on tripeptidyl derivatives have been disclosed in European Patent Applications 0 669 317, 0 686 642 and 0 648 780 and International Patent Applications WO 95/35309, WO 95/23609 and WO 94/29336.
However, there remains a need for effective inhibitors of trypsin-like serine proteases, such as thrombin. There is a particular need for compounds which are both orally bioavailable and selective in inhibiting thrombin over other serine proteases. Compounds which exhibit competitive inhibitory activity towards thrombin would be expected to be especially usefull as anticoagulants and therefore useful in the therapeutic treatment of thrombosis and related disorders.
According to the invention therm is provided a compound of formula I, 
wherein
p and q independently represent 0, 1, 2, 3 or 4;
R1 represents H, 2,3-epoxypropyl, C1-6 alkyl (which latter group is optionally substituted or terminated by one or more hydroxy group), a structual fragment of formula Ia 
wherein A1 represents a single bond or C1-4 alkylene and Rx represents H or C1-4 alkyl, provided that there are no more than six carbon atoms in the chain Rxxe2x80x94Cxe2x80x94Cxe2x80x94A1, or, when p represents 0, together with R2 represents a structural fragment of formula Ib, 
wherein
Ry represents H or C1-3 alkyl;
R2 represents H, Si(Me)3, naphthyl, indolyl, CHR21R22 or C1-4 alkyl (which latter group is optionally substituted or terminated by one or more fluorine or hydroxy group) or C3-8 cycloalkyl or phenyl (which latter two groups are optionally substituted by one or more of C1-4 alkyl, C1-4 alkoxy, halo, hydroxy, cyano, nitro, methylenedioxy, trifluoromethyl, N(H)R23, C(O)OR24), or, when p represents 0, together with R1 represents a structural fragment of formula Ib;
R3 represents H, Si(Me)3, naphthyl, indolyl, CHR25R26 or C1-6 alkyl (which latter group is optionally substitute or terminated by one or more fluorine or hydroxy group) or C3-8 cycloalkyl or phenyl (which latter two groups are optionally substituted by one or more of C1-4 alkyl, C1-4 alkoxy, halo, hydroxy, cyano, nitro, methylenedioxy, tifluoromethyl, N(H)R27 or C(O)OR28);
R21, R22, R25 and R26 independently represent cyclohexyl or phenyl;
R23 and R27 independently represent H, C1-4 alkyl or C(O)R29;
R24, R28 and Rindependently represent H or C1-4 alkyl;
R4 represents H or C1-4 alkyl;
Y represents C1-3 alkylene optionally substituted by C1-4 alkyl, hydroxy, methylene or oxo;
n represents 0, 1, 2, 3 or 4; and
B represents a structural fragment of formula IVa, IVb or IVc 
wherein
R5 represents H, halo or C1-4 alkyl; and
X1and X2 independently represent a single bond or CH2;
provided that when R1, R2 and R4 all represent H, p represents 0, Y represents (CH2)2, n represents 1 and:
(a) R3 represents unsubstituted phenyl and:
(i) B represents a structural fragment of formula IVa and R5 represents H, then q does not represent 0 or 1; and
(ii) B represents a structural fragment of formula IVb and X1 and X2 both represent CH2, then q does not represent 0; and
(b) R3 represents unsubstituted cyclohexyl, B represents a structural fragment of formula IVa and R5 represents H, then q does not represent 0;
or a pharmaceutically acceptable salt thereof (hereinafter referred to as xe2x80x9cthe compounds of the inventionxe2x80x9d).
The compounds of the invention may exhibit tautomers. All tautomexic forms and mixtures thereof are included within the scope of the invention.
The compounds of the invention may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. All diastereoisomers may be separated using conventional techniques, e.g. chromatography or factional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. frational crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric esters by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the invention.
Alkyl groups which R1, Rx, Ry, R2, R3, R4, R5, R23, R24, R27, R28 and R29 may represent and which R2, R3 and Y may be substituted by; cycloalkyl groups which R2 and R3 may represent; and alkoxy groups which R2 and R3 may be substituted by may be linear or branched, saturated or unsaturated. Alkylene groups which A1 and Y may represent may be saturated or unsaturated.
Halo groups which R5 may represent and which R2 and R3 may be substituted by include fluoro, chloro, bromo and iodo.
The wavy lines on the carbon atom in the fragments of formulae Ia, Ib, IVa, IVb and IVc signify the bond position of the fragment.
Abbreviations are listed at the end of this specification.
When B represents a structural fragment of formula IVa, IVc or IVb in which latter fragment X1 and X2 both represent CH2, preferred compounds of the invention include those wherein n represents 1.
When B represents a structural fragment of formula IVb in which X1 represents a single bond and X2 represents either a single bond or CH2, preferred compounds of the invention include those wherein n represents 2.
When B represents a structural fragment of formula IVa, preferred compounds of the invention include those wherein R5 represents H.
Preferred compounds of formula I include those wherein:
R1 represents H, methyl, 2,3-dihydroxypropyl or (2,2-dimethyl-1,3-dioxalan-4-yl)methyl;
p represents 0;
R2 represents H, optionally substituted C1-4 alkyl, or optionally substituted phenyl;
q represents 0, 1 or 2;
R3 represents C1-6 alkyl, naphthyl, indolyl, optionally substituted cyclohexyl or optionally substituted phenyl;
Y represents CH2, (CH2)2, (CH2)3, CH2CH(CH3)CH2, CH2C(xe2x95x90O)CH2 or CH2C(xe2x95x90CH2)CH2;
R4 represents H.
When R2 represents C1-4 alkyl, preferred optional substituents include hydroxy. Preferred points of attachment for the hydroxy group include the carbon atom which is xcex1 to the carbon atom to which OR1 is attached.
More preferred compounds of the invention include those wherein:
R1 represents H;
R2 represents H, methyl, hydroxymethyl or ethyl;
q represents 0;
R3 represents optionally substituted phenyl or optionally substituted cyclohexyl;
Y represents CH2, (CH2)2 or CH2C(xe2x95x90CH2)CH2;
When R1 and R2 both represent H, R3 represents unsubstituted phenyl or unsubstituted cyclohexyl and q represents 0 or 1, preferred compounds of the invention include those wherein Y rep resents CH2 or CH2C(xe2x95x90CH2)CH2.
When R1 represents H, R3 represents unsubstituted phenyl or unsubstituted cyclohexyl and q represents 0 or 1, preferred compounds of the invention include those wherein R2 represents methyl, hydroxymethyl or ethyl.
When R3 represents cyclohexyl or phenyl, preferred optional substituents include hydroxy, fluoro, chloro, methyl, methoxy, amino, nitro, trifluoromethyl, methylenedioxy, ethoxy and propoxy. Particular substituents include hydroxy, mono- or difluoro, chloro, methyl, methoxy and methylenedioxy.
Particularly preferred compounds of the invention include those wherein
Y represents CH2;
B represents a structural fragment of formula IVa.
Compounds of the invention in which the xcex1-amino acid carbon in the fragment 
is in the S-configuration are preferred. The wavy lines on the nitrogen and carbon atom in the above fragment signify the bond position of the fragment.
When R1 and R2 both represent H and p represents 0, preferred compounds of the invention are those in which the xcex1-carbon in the fragment 
is in the R-configuration. The wavy line on the carbon atom in the above fragment signifies the bond position of the fragment.
Preferred compounds of the invention include:
Ch-(R,S)CH(OH)xe2x80x94C(O)-Aze-Pab;
Ch-(R)CH(OH)xe2x80x94C(O)-Aze-Pab;
Ph-(R)CH(OH)xe2x80x94C(O)-Azn-Pab;
Ph(3-Me)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Aze-Pab;
Ph(3-OMe)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Aze-Pab;
Ph(3,5-diOMe)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Aze-Pab;
Ph(3-OMe,4-OH)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Aze-Pab;
Ph-(R,S)C(Et)(OH)xe2x80x94C(O)-Aze-Pab;
Ph-(R,S)C(Et)(OH)xe2x80x94C(O)-Pro-Pab;
(Ph)2C(OH)xe2x80x94C(O)-Aze-Pab;
Ph(3-OMe,4-OH)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Pro-Pab;
Ph-(R)CH(OH)xe2x80x94C(O)-Aze-Pac;
Ph-(R)CH(OH)xe2x80x94C(O)xe2x80x94(R,S)Pic(cis-4-Me)-Pab;
Ph(3,4-(xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94))xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Aze-Pab;
Ph(3-OMe)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Pro-Pab;
Ph(3,5-diOMe)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Pro-Pab;
Ph-(RS)C(Me)(OH)xe2x80x94C(O)-Aze-Pab;
Ph(3,5-diMe)xe2x80x94(RS)CH(OH)xe2x80x94C(O)-Aze-Pab;
Ph(3-NH2)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Aze-Pab;
Ph(3-NH2)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Pro-Pab;
Ph(3-NO2)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Pro-Pab;
Ph(3,4-(xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94))xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Pro-Pab;
Ph(3,5-diF)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Pro-Pab;
Ph-(R)CH(Oxe2x80x94CH2xe2x80x94(R,S)CH(xe2x80x94Oxe2x80x94C(CH3)2xe2x80x94Oxe2x80x94CH2xe2x80x94))xe2x80x94C(O)-Aze-Pab;
Ph-(R)C(Me)(OH)xe2x80x94C(O)-Pro-Pab;
Ph-(S)C(Me)(OH)xe2x80x94C(O)-Pro-Pab;
Ph(3,4-diF)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Pro-Pab;
Ph-(R)CH(OH)xe2x80x94C(O)xe2x80x94(R,S)Pic(4-methylene)-Pab;
Ph(3-Cl)xe2x80x94(R,S)CH(OH)xe2x80x94C(O)-Aze-Pab;
Ph-(R,S)C(xe2x80x94Oxe2x80x94C(CH3)2xe2x80x94Oxe2x80x94CH2xe2x80x94)xe2x80x94C(O)-Aze-Pab;
Ph-(R,S)C(xe2x80x94Oxe2x80x94C(CH3)2xe2x80x94Oxe2x80x94CH2xe2x80x94)xe2x80x94C(O)-Pro-Pab;
Ph-(R,S)C(CH2OH)(OH)xe2x80x94C(O)-Aze-Pab; and
Ph-(R,S)C(CH2OH)(OH)xe2x80x94C(O)-Pro-Pab.
Preparation
According to the invention there is also provided a process for the preparation of compounds of formula I which comprises:
(a) the coupling of a compound of formula V, 
wherein p, q, R1, R2 and R3 are as hereinbefore defined with a compound of formula VI, 
wherein R4, Y, n and B are as hereinbefore defined; or
(b) the coupling of a compound of formula VII, 
wherein p, q, R1, R2, R3, R4 and Y are as hereinbefore defined with a compound of formula VIII,
H2Nxe2x80x94(CH2)nxe2x80x94Bxe2x80x83xe2x80x83VIII
wherein n and B are as hereinbefore defined;
for example in the presence of a coupling system (e.g. oxalyl chloride in DMF, EDC, DCC or TBTU), an appropriate base (e.g. pyridine, DMAP or DIPEA) and a suitable organic solvent (e.g. dichloromethane, acetonitrile or DMF).
Compounds of formula V are either commercially available, are well known in the literature, or are available using known techniques.
For example, compounds of formula V wherein R1 and R2 both represent H, p and q both represent 0 and R3 represents naphthyl or phenyl optionally substituted by one or more of C1-4 alkyl, C1-4 alkoxy, halogen, hydroxy, cyano, methylenedioxy, nitro, trifluoromethyl, N(H)R27 or C(O)OR28 may be prepared by reaction of an aldehyde of formula IX,
R3aCHOxe2x80x83xe2x80x83IX
wherein R3a represents naphthyl or phenyl optionally substituted by one or more of C1-4 alkyl, C1-4 alkoxy, halogen, hydroxy, cyano, methylenedioxy, nitro, trifluoromethyl, N(H)R27 or C(O)OR28 and R27 and R28 are as hereinbefore defined, with:
(i) a compound of formula X
Rxe2x80x3CNxe2x80x83xe2x80x83X
wherein Rxe2x80x3 represents H or (CH3)3Si, for example at elevated temperature (e.g. above room temperature but below 100xc2x0 C.) in the presence of a suitable organic solvent (e.g. chloroform) and, if necessary, in the presence of a suitable catalyst system (e.g. benzylammonium chloride), followed by hydrolysis in the presence of an appropriate base (e.g. NaOH);
(ii) chloroform, for example at elevated temperature (e.g. above room temperature but below 100xc2x0 C.) in the presence of a suitable organic solvent (e.g. chloroform) and, if necessary, in the presence of a suitable catalyst system (e.g. benzylammonium chloride), followed by hydrolysis in the presence of an appropriate base (e.g. NaOH);
(iii) a compound of formula XI, 
wherein M represents Mg or Li, followed by oxidative cleavage (e.g. ozonolysis or osmium or ruthenium catalysed) under conditions which are well known to those skilled in the art; or
(iv) tris(methylthio)methane under conditions which are well known to those skilled in the art, followed by hydrolysis in the presence of an appropriate base.
Compounds of formula V wherein R1 represents H, R2 represents CH2OH, p and q both represent 0 and R3 represents naphthyl or phenyl optionally substituted by one or more of C1-4 alkyl C1-4 alkoxy, halogen, hydroxy, cyano, methylenedioxy, nitro, trifluoromethyl, N(H)R27 or C(O)OR28 may be prepared by reaction of a compound of formula XII,
R3aC(O)C2H5xe2x80x83xe2x80x83XII
wherein R3a is as hereinbefore defined with sodium hypochorite for example at room temperature in the presence of a suitable solvent (e.g. water).
Compounds of formula VI and VII are either commercially available, are well known in the literature, or are available using known techniques. For example compounds of formula VI may be made by standard peptide coupling of a compound of formula XIII, 
wherein R4 and Y are as hereinbefore defined with a compound of formula VIII as hereinbefore defined for example under conditions such as those described hereinbefore for synthesis of compounds of formula I. Similarly compound of formula VII may also be made by standard peptide coupling of a compound of formula XIII as hereinbefore defined with a compound of formula V as hereinbefore defined for example under conditions such as those described hereinbefore for synthesis of compounds of formula I.
Compounds of formula VIII, IX, X, XI, XII and XIII are either commercially available, are well known in the literature, or are available using known techniques. Substituents on the phenyl group in compounds of formula V, VII, IX and XII may be interconverted by techniques well known to those skilled in the art.
The compounds of the invention may be isolated from their reaction mixtures using conventional techniques.
It will be appreciated by those skilled in the art that in the process described above the functional groups of intermediate compounds may need to be protected by protecting groups.
Functional groups which it is desirable to protect include hydroxy, amino, amidino, guanidino and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl and diarylalkylsilyl groups (e.g. tertbutyldimethylsilyl, tertbutyldiphenylsilyl or trimethylsilyl) and tetrahydropyranyl. Suitable protecting groups for hydroxy groups, which groups are attached to adjacent carbon atoms include O,Oxe2x80x2-isopropylidene. Suitable protecting groups for amino, amidino and guanidino include tertbutyloxycarbonyl or benzyloxycaronyl. Amidino and guanidino nitrogens may be either mono- or diprotected. Suitable protecting groups for carboxylic acid include C1-6 alkyl or benzyl esters.
The protection and deprotection of functional groups may take place before or after coupling.
In particular, the compounds of the invention may be prepared by processes comprising the coupling of an N-acylated amino acid or an N-protected amino acid. When an N-protected amino acid is used the acyl group may be added after coupling and deprotection of the nitrogen atom may then be effected using standard methods thereafter.
Protecting groups may be removed in accordance with techniques which are well known to those skilled in the art and as described hereinafter.
Certain protected intermediates of formula I, in which the amidino and guanidino nitrogens in B are protected, and which may be made prior to a final deprotection stage to form compounds of the invention, are novel.
According to a further aspect of the invention there is provided a compound of formula XIV, 
wherein B1 represents a structural fragment of formula IVd, IVe or IVf 
D1 and D2 independently represent H or benzyloxycarbonyl and p, q, R1, R2, R3, R4, Y, n, R5,
X1 and X2 are as hereinbefore defined, provided that D1 and D2 do not both represent H.
The wavy lines on the carbon atom in the fragments of formulae IVd, IVe or IVf signify the bond position of the fragment.
The use of protecting groups is fully described in xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, edited by J W F McOmie, Plenum Press (1973), and xe2x80x9cProtective Groups in Organic Synthesixe2x80x9d, 2nd edition, T W Greene and P G M Wutz, Wiley-Interscience (1991).
It will also be appreciated by those skilled in the art that, although such protected derivatives of compounds of formula I may not possess pharmacological activity as such, they may be administered parenterally or orally and thereafter metabolised in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as xe2x80x9cprodrugsxe2x80x9d. All prodrugs of compounds of formula I are included within the scope of the invention.
Protected derivatives of compounds of formula I which are particularly usefull as prodrugs include compounds of formula XIV.
Medical and Pharmaceutical Use
The compounds of the invention are useful because they possess pharmacological activity. They are therefore indicated as pharmaceuticals.
According to a further aspect of the invention there is thus provided the compounds of the invention for use as pharmaceuticals.
In particular, the compounds of the invention are potent inhibitors of thrombin, for example as demonstrated in the tests described below.
The compounds of the invention are thus expected to be usefull in those conditions where inhibition of thrombin is required.
The compounds of the invention are thus indicated in the treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues of animals including man.
The compounds of the invention are further indicated in the treatment of conditions where there is an undesirable excess of thrombin without signs of hyperagulabity, for example in neurodegenerative diseases such as Alizheimer""s disease.
Particular disease states which may be mentioned include the treatment of and/or prophylaxis of venous thrombosis and pulmonary embolism arterial thrombosis (e.g. in myocardial infarction unstable angina, thrombosis-based stroke and peripheral arterial thrombosis) and systemic embolism usually from the atrium during arterial fibrillation or from the left ventricle after trarsmural myocardial infarction.
Moreover, the compounds of the invention are expected to have utility in prophylaxis of re-occlusion (i.e. thrombosis) after thrombolysis, percutaneous tansluminal angioplasty (PTCA), coronary bypass operations, microsurgery and vascular surgery in general.
Further indications include the treatment and prophylaxis of disseminated intravascular coagulation caused by bacteria, multiple trauma, intoxication or any other mechanism; anticoagulant treatment when blood is in contact with foreign surfaces in the body such as vascular grafts, vascular stents, vascular catheters, mechanical and biological prosthetic valves or any other medical device; and anticoagulant treatment when blood is in contact with medical devices outside the body such as during cardiovascular surgery using a heart-lung machine or in haemodialysis.
In addition to its effects on the coagulation process, thrombin is known to activate a large number of cells (such as neutrophils, fibroblasts, endothelial cells and smooth muscle cells). Therefore, the compounds of the present invention may also be usefull for the treatment or prophylaxis of idiopathic and adult respiratory is syndrome, pulmonary fibrosis following treatment with radiation or chemotherapy, septic shock, septicemia, inflammatory responses, which include, but are not limited to, edema, acute or chronic atherosclerosis such as coronary arterial disease, cerebral arterial disease, peripheral arterial disease, reperfusion damage, and restenosis after percutaneous trans-luminal angioplasty (PTCA).
Compounds of the present invention that inhibit trypsin and/or thrombin may also be useful in the treatment of pancreatic.
According to a further aspect of the invention, there is provided a method of treatment of a condition where inhibition of thrombin is required which method comprises administration of a therapeutically effective amount of a compound of formula I as defined above, or a pharmaceutically acceptable salt thereof, to a person suffering from, or susceptible to, such a condition.
Pharmaceutical Preparations
The compounds of the invention will normally be administered orally, subcutaneously buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route or via inhalation, in the form of pharmaceutical preparations comprising the active ingredient either as a free base, or a pharmaceutical acceptable non-toxic organic or inorganic acid addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.
The compounds of the invention may also be combined with any antithrombotic agent with a different mechanism of action, such as the antiplatelet agents acetylsalicylic acid, ticlopidine, clopidogrel, thromboxane receptor and/or synthetase inhibitors, fibrinogen receptor antagonists, prostacyclin mimetics and phosphodiestease inhibitors.
The compounds of the invention may further be combined with thrombolytics such as tissue plasminogen activator (natural or recombinant), streptokinase, uroknase, prouroknase, anisolated streptokinase plasminogen activator complex (ASPAC), animal salivary gland plasminogen activators, and the like, in the treatment of thrombotic diseases, in particular myocardial infarction.
According to a further aspect of the invention there is thus provided a pharmaceutical formulation including a compound of formula I as hereinbefore defined, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
Suitable daily doses of the compounds of the invention in therapeutical treatment of humans are about 0.001-100 mg/kg body weight at peroral administration and 0.001-50 mg/kg body weight at parenteral administration.
The compounds of the invention have the advantage that they may be more efficacious, be less toxic, be longer acting, have a broader range of activity, be more potent, produce fewer side effects, be more easily absorbed than, or that they may have other usefull pharmacological properties over, compounds known in the prior art.
Biological Tests
Test A
Determination of Thrombin Clotting Time (TT)
Human thrombin (T 6769, Sigma Chem Co) in buffer solution, pH 7.4, 100 xcexcl, and inhibitor solution, 100 xcexcl, were incubated for one min. Pooled normal citrated human plasma, 100 xcexcl, was then added and the clotting time measured in an automatic device (KC 10, Amelung).
The clotting time in seconds was plotted against the inhibitor concentration, and the IC50TT was determined by interpolation.
IC50TT is the concentration of inhibitor that doubles the thrombin clotting time for human plasma.
Test B
Determination of Activated Partial Thromboplastin Time (APTT)
APTT was determined in pooled normal human citrated plasma with the reagent PTT Automated 5 manufactured by Stago. The inhibitors were added to the plasma (10 xcexcl inhibitor solution to 90 xcexcl plasma) followed by the reagent and calcium chloride solution and APTT was determined in the mixture by use of the coagulation analyser KC10 (Amelung) according; to the instructions of the reagent producer. The clotting time in seconds was plotted against the inhibitor concentration in plasma and the IC50APTT was determined by interpolation. IC50APTT is defined as the concentration of inhibitor in human plasma that doubled the Activated Partial Thromboplastin Time.
Test C
Determination of Thrombin Time ex vivo
The inhibition of thrombin after oral or parenteral administration of the compounds of the invention were examined in conscious rats which, one or two days prior to the experiment, were equipped with a catheter for blood sampling from the carotid artery. On the experimental day blood samples were withdrawn at fixed times after the administration of the compound into plastic tubes containing 1 part sodium citrate solution (0.13 mol per L) and 9 parts of blood. The tubes were centriflged to obtain platelet poor plasma The plasma was used for determination of thrombin time as described below.
The citrated rat plasma, 100 xcexcL, was diluted with a saline solution, 0.9%, 100 xcexcL, and plasma coagulation was started by the addition of human thrombin (T 6769, Sigma Chem Co, USA) in a buffer solution, pH 7.4, 100 xcexcL. The clotting time was measured in an automatic device (KC 10, Amelumg, Germany).
Where a compound of formula XIV was administered, concentrations of the appropriate active thrombin inhibitor of formula I in the rat plasma were estimated by the use of standard curves relating the thrombin time in the pooled citrated rat plasma to known concentrations of the corresponding xe2x80x9cactivexe2x80x9d thrombin inhibitor dissolved in saline.
Test D
Determination of Thrombin Time in Urine ex vivo
Conscious rats were placed in metabolism cages for 24 hours following oral administration of compounds of the invention. The thrombin time was determined on the collected urine as described below.
Pooled normal citrated human plasma (100 xcexcL) was incubated with the concentrated rat urine, or saline dilutions thereof, for one minute. Plasma coagulation was then initiated by the administration of human thrombin (T 6769, Sigma Chem Company) in buffer solution (pH 7.4; 100 xcexcL). The clotting time was measured in an automatic device (KC 10; Amelung).
Where a compound of formula XIV was administered, concentrations of the appropriate active thrombin inhibitor of formula I in the rat urine were estimated by the use of standard curves relating the thrombin time in the pooled normal citrated human plasma to known concentrations of the corresponding xe2x80x9cactivexe2x80x9d thrombin inhibitor dissolved in concentrated rat urine (or saline dilutions thereof). By multiplying the total rat urine production over the 24 hour period with the estimated mean concentration of the aforementioned active inhibitor in the urine, the amount of the active inhibitor excreted could be calculated.